Essential Cell Biology 5th edition

362 CHAPTER 10 Analyzing the Structure and Function of Genes
KEY TERMS
complementary DNA (cDNA)
cDNA library
CRISPR
dideoxy (Sanger) sequencing
DNA cloning
DNA library
DNA ligase
gene knockout
genomic library
green fluorescent protein
(GFP)
hybridization
in situ hybridization
plasmid
polymerase chain reaction (PCR)
recombinant DNA
reporter gene
restriction enzyme
RNA interference (RNAi)
RNA-Seq
transformation
transgenic organism
ESSENTIAL CONCEPTS
• DNA technology has revolutionized the study of cells, making it possible
to pick out any gene at will from the thousands of genes in a cell
and to determine its nucleotide sequence.
• A crucial element in this technology is the ability to cut a large DNA
molecule into a specific and reproducible set of DNA fragments using
restriction enzymes, each of which cuts the DNA double helix only at
a particular nucleotide sequence.
• DNA fragments can be separated from one another on the basis of
size by gel electrophoresis.
• DNA cloning techniques enable any DNA sequence to be selected
from millions of other sequences and produced in unlimited amounts
in pure form.
• DNA fragments can be joined together in vitro by using DNA ligase to
form recombinant DNA molecules that are not found in nature.
• DNA fragments can be maintained and amplified by inserting them
into a larger DNA molecule capable of replication, such as a plasmid.
This recombinant DNA molecule is then introduced into a rapidly
dividing host cell, usually a bacterium, so that the DNA is replicated
at each cell division.
• A collection of cloned fragments of chromosomal DNA representing
the complete genome of an organism is known as a genomic library.
The library is often maintained as millions of clones of bacteria,
each different clone carrying a different fragment of the organism’s
genome.
• cDNA libraries contain cloned DNA copies of the total mRNA of a
particular type of cell or tissue. Unlike genomic DNA clones, cDNA
clones contain predominantly protein-coding sequences; they lack
introns, regulatory DNA sequences, and promoters. Thus they are
useful when the cloned gene is needed to make a protein.
• Nucleic acid hybridization can detect any given DNA or RNA sequence
in a mixture of nucleic acid fragments. This technique depends on
highly specific base-pairing between a labeled, single-stranded
DNA or RNA probe and another nucleic acid with a complementary
sequence.
• The polymerase chain reaction (PCR) is a powerful form of DNA
amplification that is carried out in vitro using a purified DNA polymerase.
Cloning via PCR requires prior knowledge of the sequence to
be amplified, because two synthetic oligonucleotide primers must be
synthesized that bracket the portion of DNA to be replicated.